1. Field of the Invention
This invention relates generally to polyethylene glycol derivatives (PEGs), their fabrication and use, and more particularly to their use in aqueous environments and even more particularly to their use in aqueous environments to modify other substances such as proteins or surfaces.
2. Description of the Background
PEGs are neutral, hydrophilic polymers that are also soluble in water and a variety of organic solvents, and which possess a wide array of biomedical and biotechnical applications. Once in aqueous solution they are heavily hydrated, highly mobile and exclude other polymers (including proteins and nucleic acids). As a consequence PEGs are nontoxic, nonimmunogenic, and capable of forming aqueous two-phase systems with a variety of polymers. Molecules coupled to PEG become nontoxic, nonimmunogenic, soluble in water and many organic solvents, and surfaces modified by PEG attachment become hydrophilic and protein rejecting. These properties have led to a variety of biotechnical and biomedical applications including: aqueous two-phase partitioning, protein (i.e., enzyme, antibody, antigen) immobilization, drug modification, and preparation of protein rejecting surfaces. In addition, PEG-coated surfaces can be used to control wetting and electroosmosis. The properties of PEG mentioned above are described in the following papers which are hereby incorporated by reference: B. J. Herren, S. G. Slater, J. M. Van Alstine, J. M. Harris, and R. S. Snyder, J. Colloid Interface Sci., 51, 46-55 (1987); and J. M. Harris, D. E. Brooks, J. F. Boyce, R. S. Snyder, and J. M. Van Alstine, in "Dynamic Aspects of Polymer Science" J. D. Andrade, Ed., Plenum 1988 pp. 111-119.
Preparation of new activated PEGs has become central to many studies on PEG applications as shown by J. M. Harris in J. Macromol. Sci.-Rev. Macromol. Chem. Phys., C25, 325-373 (1983) which is hereby incorporated by reference. Typically, useful PEGs contain at least one electrophilic center available for reaction with nucleophilic centers of biomolecules (e.g., lysine, cysteine and like residues of proteins) or surfaces (e.g., aminated glass). A variety of active PEGs have been prepared, yet there is continued interest in synthesis of new derivatives possessing properties not now available. A property of particular interest is increased selectivity, including relative inertness toward water and high reactivity toward desired functional groups. As an example, PEG tresylate, a much used derivative, is reactive toward a variety of nucleophiles including water, while PEG aldehydes are inert toward water and react primarily with amines. Inertness toward water is desired, not only because of efficiency of storage, preparation, and application, but also because it permits stepwise linkage, in aqueous media, of molecules to surfaces and molecules to molecules.
Preparation of activated and selective PEGs mentioned above are provided in the following references which are hereby incorporated by reference: K. Yoshinaga and J. M. Harris, J. Bioact. Comp. Polym., 1, 17-24 (1989); K. Nilsson and K. Mosbach, Methods in Enzymology, 104, 56 (1984); C. Delgado, G. E. Francis, and D. Fisher, in "Separations Using Aqueous Phase Systems," D. Fisher and I. A. Sutherland, Eds., Plenum, London, 1989, pp. 211-213; M.-B. Stark and J. K. Holmberg, Biotech. Bioeng., 34, 942 (1989); J. M. Harris and K. Yoshinaga, J. Bioact. Compat. Polym., 4, 281 (1989); H. Walter, D. E. Brooks, and D. Fisher (Editors), "Partitioning in Aqueous Two-Phase Systems," Academic Press, Orlando, Fla., 1985; D. Fisher and I. A. Sutherland (Editors), "Separations Using Aqueous Phase Systems: Applications in Cell Biology and Biotechnology," Plenum, London, 1989.
U.S. Pat. No. 4,002,531 (1977) describes a preparation of PEG acetaldehyde for attaching PEG to enzymes and other proteins. However, the subsequently published article by Harris mentioned above, indicates that the preparation as described in the U.S. Pat. No. 4,002,531 gives very little of the desired aldehyde. More importantly, PEG acetaldehyde is unstable in the presence of base and of limited utility for protein or surface modification in water as shown by M. S. Paley and J. M. Harris, in J. Polym Sci. Polym Chem. Edn., 25, 2447-2454 (1987).
U.S. Pat. No. 4,179,337 (1979) to Davis et al indicates that PEG can be attached to proteins (such as enzymes and insulin) to provide PEG-protein conjugates which are soluble and nonimmunogenic. Several uses for these nonimmunogenic PEG-proteins are described and claimed. Also a wide variety of methods for attaching PEG to proteins to provide soluble PEG-protein conjugates are described and claimed. Each of the Davis et al methods for attaching PEG to proteins uses conventional, water-sensitive PEGs.